Nontumorigenic mutants of Agrobacterium tumefaciens will be isolated following treatment with nitrosoguanidine. Mutants will be characterized with respect to growth requirements, bacteriophage sensitivity and presence or absence of the Ti plasmid. Among those mutants still harboring the Ti plasmid, genetic technicians will be used to determine whether the mutation effects plasmid or chromosomal genes. Plasmid mutations will be physically mapped using heteroduplex formation followed by Sl and restriction endonuclease cleavage. Such mutations will be compared with Ti plasmid deletion mutations isolated by use of transposons. Chromosomal mutations will be genetically analyzed using chromosomal mobilization techniques developed in this laboratory. Such mutations will be mapped with respect to nutritional markers. In such a manner the number, location and linkage state of genes controlling tumorigenesis can be determined. In addition, other Agrobacterium plasmids, bearing no relationships to the Ti plasmids will be screened for sex factor activity. If such elements can be found, an interrupted mating system will be developed.